The research proposed in this application continues a long-standing interest in the mechanisms by which nuclearly coded mitochondrial proteins are targeted, translocated, posttranslocationally processed, folded, and assembled into their native, active structures. These processes are central to the maintenance and propagation of mitochondria, and, hence, to cellular metabolism and homeostasis. A thorough knowledge and understanding of these pathways is critical to illuminating the pathogenesis of human metabolic disease involving mitochondrial enzymes and to the rational design of strategies for somatic correction of their deficiencies. The model system used for these studies is intact rat liver mitochondria and subfractions or components purified therefrom, and the model substrate is mammalian ornithine transcarbamylase (OTC), a liver-specific, mitochondrial enzyme deficient in hyperammonemia. The specific aims include: i) characterization of the mitochondria processing peptidase (MPP), one of the enzymes responsible for the posttranslocational processing of OTC by overexpressing it in E. coli, defining its active site and metal ion requirements, disrupting its activity by site-directed mutagenesis, crystallizing it, and solving its structure; 2) identifying, purifying, and characterizing other mitochondrial proteases required to cleave a subset of imported precursors; 3) isolating and cloning leader peptide receptors and components of the translocation apparatus (contact site) by recovering translocation complexes, cross-linking translocation intermediates to members of the apparatus, and purifying identified proteins immunochemically; 4) exploring the role of individual amino acid residues in the chaperonin groEL (homologous to the rat Hsp60 protein) in ATP hydrolysis, substrate protein binding, groES activation, and protein folding; and 5) determining whether multimeric enzyme or complex assembly is a protein. mediated process and what is the nature and identity of the factors required for this function.